Bi technology IranianJournal of

Background: The High Resolution Melting (HRM) method is a new scanning method for detecting unknown changes in DNA and its advantages have persuaded researchers to recruit it as a screening method. Objectives: Here, we developed a HRM method to screen R188H SNP (rs3218536) of XRCC2 and compared the results with a well known PCR-RFLP technique. Materials and Methods: Genomic DNA samples from 350 healthy individuals were obtained. PCR-HRM analysis and PCR-RFLP method were performed simultaneously. Results: Three different melting profiles corresponding to three different genotypes recognized by HRM analysis. The results of PCR-RFLP showed no discrepancy. Conclusions: We concluded that the HRM technique can be used as a screening method for rapid discrimination of R188H genotypes in XRCC2 gene

MicroRNA 138 Upregulation is Associated with Decreasing Levels of CCND1 Gene Expression and Promoting Cell Death in Human Prostate Cancer Cell Lines

Background: This research intended to discover the significance of miR-138 (microRNA 138) on the expression profile, proliferation, and the associated regulatory mechanisms in prostate cancer (PCa). Methods: Thirty-five specimens of prostate were studied to evaluate the expression level of miR138 by RT-qPCR (Quantitative reverse transcription polymerase chain reaction). Bioinformatics analysis was performed to search for the target genes of miR-138; and ABL1 (ABL proto-oncogene 1, non-receptor tyrosine kinase), CCND1 (cyclin D1), CCND3 (cyclin D3), VIM (vimentin), TWIST1 (twist family bHLH transcription factor 1), HIF1A (hypoxia-inducible factor 1 subunit alpha), and TERT (telomerase reverse transcriptase) genes were selected. Then, the biological role of miR-138 and CCND1 in the progression of PCa was investigated using RT-qPCR and luciferase reporter gene assay. Finally, overexpression of miR-138 on the proliferation in PCa cell lines was analyzed using the MTT (3-(4, 5-dimethylthiazol-2-Yl)-2, 5-diphenyltetrazolium bromide, Sigma, Germany) assay. Results: RT-qPCR showed that the expression of miR-138 downregulated in PCa tissues and cell lines. Bioinformatics analysis and RT-qPCR assay demonstrated that CCND1 expression level was negatively correlated with miR-138 in PCa tissues and the PC3 cell line. Moreover, CCND1 was predicted to be the target gene of miR138 in the PC3 cell line based on the results of luciferase reporter gene assay. Substantially, over-expression of miR138-5p mimic could inhibit the expression level of CCND1 gene in PC3 cell lines. Lastly, over-expression of miR-138 inhibited the proliferative capacities in PC3 and DU-145 cells. Conclusion: Our research introduces miR-138 as a negative regulator of CCND1 in the progression of PCa with an inhibitory impact on the proliferation rate of PCa cell lines. This regulatory mechanism could be utilized for the design and target selection of remedial miRNA-based approaches

Optimization and Comparison of the PolyFect Gene Delivery Method in Three Different Kinds of Mesenchymal Stem Cell Types

Objective: The aim of this study was optimization of the PolyFect gene delivery methodof pcDNA3.1 expression vector transfected with the mouse pdx-1 gene in three differentkinds of mesenchymal stem cells and Hepa cells as well as comparison of transfectionefficiency leading to expression of the mentioned gene in the cell types used.Materials and Methods: Rat bone marrow-derived mesenchymal stem cells, C57 mousebone marrow-derived mesenchymal stem cells, human synovium derived mesenchymalstem cells and Hepa cells were used in this study. After culturing of the mentioned cells,mouse pdx-1 gene were transfected into them using the Qiagen PolyFect kit. 72 hourslater, the cells were treated with anti-mouse Pdx-1 antibody and immunocytochemicallyanalyzed using a fluorescent inverted microscope. Transfection conditions were optimizedin each of these cells by changing different lipofection parameters such as DNAconcentration, PolyFect reagent concentration and cell density.Results: The results demonstrated that for transfection of these cells, the best concentrationsof DNA and PolyFect reagent are 400 ng/μL and 6000 ng/μL respectively. For maximumtransfection efficiency, the best cell density in 12-well plates was 105 cells in Hepacells, 1.3×105 cells in rat bone marrow-derived mesenchymal stem cells, 1.5×105 cells inhuman synovium-derived mesenchymal stem cells and 105 cells in C57 mouse bone marrow-derived mesenchymal stem cells. Under the mentioned optimized conditions, the maximumefficiency of transfection was determined to be 50% for Hepa cells, 40% for rat bonemarrow-derived mesenchymal stem cells, 21% for human synovium-derived mesenchymalstem cells and 10% for C57 mouse bone marrow-derived mesenchymal stem cells.Conclusion: These findings implicate that the most important factor extremely influencingtransfection efficiency in mesenchymal stem cells is the cell derivation origin. Resultsof this study can be used in basic and clinical studies dealing with gene therapy in mesenchymalstem cells

Assessment of genetic mutations in the XRCC2 coding region by high resolution melting curve analysis and the risk of differentiated thyroid carcinoma in Iran

Homologous recombination (HR) is the major pathway for repairing double strand breaks (DSBs) in eukaryotes and XRCC2 is an essential component of the HR repair machinery. To evaluate the potential role of mutations in gene repair by HR in individuals susceptible to differentiated thyroid carcinoma (DTC) we used high resolution melting (HRM) analysis, a recently introduced method for detecting mutations, to examine the entire XRCC2 coding region in an Iranian population. HRM analysis was used to screen for mutations in three XRCC2 coding regions in 50 patients and 50 controls. There was no variation in the HRM curves obtained from the analysis of exons 1 and 2 in the case and control groups. In exon 3, an Arg188His polymorphism (rs3218536) was detected as a new melting curve group (OR: 1.46; 95%CI: 0.432-4.969; p = 0.38) compared with the normal melting curve. We also found a new Ser150Arg polymorphism in exon 3 of the control group. These findings suggest that genetic variations in the XRCC2 coding region have no potential effects on susceptibility to DTC. However, further studies with larger populations are required to confirm this conclusion

Changes in salivary gland function following radioiodine therapy of thyroid diseases: A comparison of high-dose therapy for thyroid cancer and low-dose therapy for benign thyroid disease

ABSTRACT Introduction: High-dose radioactive iodine therapy in differentiated thyroid cancer (DTC) may adversely affect the salivary gland function. This study is aimed to evaluate the effect of radioactive iodine (RAI) with dose of 100 mCi in DTC patients compared to lower doses of less than 30 mCi in hyperthyroid cases. Methods: Fifty four patients (13 men and 41 women) age: 42.3±14.3 (21-71) years were enrolled in the study. Twenty seven hyperthyroid cases received less than 30 mCi of I-131 for the treatment, and 27 DTC patients were treated with 100 mCi of I-131. All patients underwent Tc-99m pertechnetate scintigraphy before and three months after radioiodine therapy. Salivary gland excretion fractions (EF) were compared between groups. A decrease of more than 5% in EF was considered significant. Results: The total frequency of salivary dysfunction was 41.7%. In patients received a dosage of 100 mCi of I-131, this frequency was 49.1%, while with less than 30 mCi, it was 34.3% (p<0.01). The right parotid gland was affected more than the left following 100 mCi treatment. Risk ratio of salivary gland involvement in high-dose versus low-dose group was significant (risk ratio=1.04-1.98, CI (95%); p=0.019). However, there was no significant difference in symptom presentation, i.e. xerostomia between two groups. Conclusion: RAI therapy may cause salivary gland dysfunction and this effect is more frequent in DTC patients with higher dose of 100 mCi compared to hyperthyroid cases with lower doses of less than 30 mCi

Co-delivery of PLGA nanoparticles loaded with rSAG1 antigen and TLR ligands: An efficient vaccine against chronic toxoplasmosis

International audienceAlthough vaccination is a promising approach for the control of toxoplasmosis, there is currently no commercially available human vaccine. Adjuvants such as delivery vehicles and immunomodulators are critical components of vaccine formulations. In this study, Poly (D, l-lactide-co-glycolide) (PLGA) nanoparticles were applied to serve as delivery system for both surface antigen-1 (SAG1), a candidate vaccine against toxoplasmosis and two TLR ligands, monophosphoryl lipid A (MPL) and imiquimod (IMQ), respectively. Compared to rSAG1 alone, CBA/J mice immunized with rSAG1-PLGA produced higher anti-SAG1 IgG antibodies titers. This response was increased by the co-administration of IMQ-PLGA (p < 0.01). Compared to IMQ-PLGA co-administration, MPL-PLGA co-administration further increased the humoral response (p < 0.01) and potentiated the Th1 humoral response. Compared to rSAG1 alone, rSAG1-PLGA, or rSAG1-PLGA mixed with IMQ-PLGA or MPL-PLGA similarly enhanced the cellular response characterized by the production of IFN-γ, IL-2, TNF-α and low levels of IL-5, indicating a Th1-biased immunity. The induced immune responses, led to significant brain cyst reductions (p < 0.01) after oral challenge with T. gondii cysts in mice immunized with either rSAG1-PLGA, rSAG1-PLGA + IMQ-PLGA, rSAG1-PLGA + MPL-PLGA formulations. Taken together the results indicated that PLGA nanoparticles could serve as a platform for dual-delivery of antigens and immunomodulators to provide efficacious vaccines against toxoplasmosis

E23K POLYMORPHISM IN IRANIAN PATIENTS WITH CORONARY HEART DISEASE

Abstract &nbsp;&nbsp; BACKGROUND: It has been shown that ATP-sensitive potassium (KATP) channels play an important role in physiology of myocardial adaptation to ischemia. In cardiomyocytes, the pore-forming subunits of these channels are coded by KCNJ11 gene. It was reported that the common polymorphism E23K of this gene is associated with higher susceptibility to coronary heart disease (CHD) in Chinese patients, but no other reports are available from other ethnic groups. &nbsp;&nbsp; METHODS: Iranian patients with confirmed CHD, aged over 50 years were compared with healthy controls for allelic and genotypic frequencies of this polymorphism. Patients who did not suffer from diabetes mellitus were entered into this study if they showed coronary stenosis of &gt;&nbsp;50% in at least one artery in the angiography performed after a coronary event. The subjects and controls were matched for age, gender, blood glucose, body mass index and smoking. &nbsp;&nbsp; RESULTS: No association could be found between CHD and frequencies of G and A alleles, single genotype frequencies of AA, AG, GG, and combine genotypes frequencies of AG+AA versus GG, and AG+GG versus AA. &nbsp;&nbsp; CONCLUSION: This study did not find any association between coronary heart disease and E23K polymorphism in Iranian patients. But, this finding is not conclusive due to limitation of sample size. A subsequent study with a larger sample size is recommended. &nbsp; &nbsp;&nbsp; Keywords: Single Nucleotide Polymorphism, Kir6.2 channel, KATP Channels, Caucasian. &nbsp;</p

HETEROLOGOUS PRODUCTION OF DENSE GRANULE GRA7 ANTIGEN OF TOXOPLASMA GONDII IN ESCHERICHIA COLI

Abstract. Infection with Toxoplasma gondii causes serious health problems in congenitally-infected and immunocompromised individuals. Numerous studies have shown usefulness of dense granule antigens of T. gondii in serodiagnosis of the infection and induction of protective immunity. This study describes cloning, expression, purification and antigenicity evaluation of recombinant GRA7 protein (rGRA7). DNA encoding GRA7, amino acids 18 to 236, was obtained from Toxoplasma gondii RH strain by polymerase chain reaction amplification and cloned in prokaryotic expression plasmid pET-28b(+). Sequence analysis showed 97% similarity between GRA7 gene fragment and published sequence of gra7. Recombinant protein was expressed in Escherichia coli and purified in a single step by immobilized metal ion affinity chromatography. Antigenicity of the protein was evaluated in Western blot analysis showing human sera from acute T. gondii infection strongly reacted with rGRA7 while sera from chronic infection weakly recognized the protein. Negative sera failed to react with rGRA7. The antigenic rGRA7 might be used, in combination with other T. gondii antigen, to develop more efficacious diagnostic tests and/or in vaccine formulations